WO2007014974A1

WO2007014974A1 - Aseptic mycorrhization inoculant and in vitro and ex vitro application methods

Info

Publication number: WO2007014974A1
Application number: PCT/ES2006/070115
Authority: WO
Inventors: Custodia Purificación CANO ROMERO; Alberto Bago Pastor
Original assignee: Consejo Superior De Investigaciones Científicas
Priority date: 2005-07-29
Filing date: 2006-07-27
Publication date: 2007-02-08
Also published as: ES2268984B1; ES2268984A1

Abstract

The invention relates to an aseptic mycorrhization inoculant which is produced in vitro, which is packed and distributed under sterile conditions and which, as such, is suitable for the mycorrhization of plants both in vitro (for use in the nursery production of plants, particularly in the case of micropropagated plants) and ex vitro (for use in the production of plants in pots and seedling nurseries, as well for use on farms and in soil revegatation programmes). For said purpose, a low-viscosity, light, semi-solid agar-type base culture medium is used to facilitate the penetration of the root in the inoculant, as well as to potentiate and preserve the infectiousness of arbuscular mycorrhizal fungi (AMF). The inventive inoculant has many advantages over commercial inoculants already on the market in that it can be applied more easily, quickly and cleanly, it produces immediate effects and it can be used directly in in vitro cultures and enables continuous asepsis certification until the moment of application thereof.

Description

TITLE

ASEPTIC MICORRIZATION INOCULANT, AND PROCEDURES FOR

APPLICATION IN CONDITIONS IN VITROY EX VITRO.

SECTOR OF THE TECHNIQUE

Organic farming, nursery production, revegetation. Natural biofertilizer for most plants of economic and ecological interest (horticultural, ornamental, used for revegetation, etc.) that increases their survival, vitality and production, and reduces the need for chemical fertilizers, pesticides and phytosanitary applications.

STATE OF THE TECHNIQUE

Agricultural practices used in developed countries have tended, in recent years, towards the intensive exploitation of available resources (eg localized irrigation, crops under plastic, etc.) with a view to optimizing yields minimizing both economic and environmental costs ( López-Gorgé, 1991). The risk of overexploitation derived from these intensive practices has given the alarm against the negative impact on the quality of soils and natural aquifers has the massive and often indiscriminate use of chemical fertilizers and phytosanitary products. That is why the so-called "sustainable agriculture" (Harwood, 1991), which is integrated into a broader concept, is called "sustainable ecosystems" (Jeffries and Barea, 2001). "Sustainability" could be defined as the art of successfully managing the available natural resources in a way that meets social needs, while maintaining or increasing the quality of the natural environment and the conservation of its resources (Gianinazzi and Schüepp, 1994 ). The continuous increase in environmental degradation and instability, and particularly in the fragility of natural and agricultural soils, are promoting a progressive awareness of the need to develop more rational and sustainable use practices of agroecosystems. It is precisely in these situations that the fundamental importance of rhizospheric microorganisms in general, and of mutualistic symbiotics in in particular, as natural stabilizers of soils. Among these microorganisms, arbuscular mycorrhizal fungi (AMF) stand out, obligate symbionts common inhabitants of the soils that establish mutualistic associations with the roots of more than 80% of the plants, and whose importance in the nutritional improvement of the plants, as well as In overcoming them by all types of biotic and abiotic stresses is widely documented (Smith and Read, 1997). These beneficial effects are especially noticeable for almost all plants of horticultural, ornamental interest, as well as for plants used in revegetation and recovery programs of degraded soils.

Theoretically, the extensive use of AMF as natural biofertilizers would be of great interest, since it would greatly improve the development and production of plants with minimal agrochemical amendments (Mosse, 1986). In fact, HMAs have demonstrated their effectiveness by increasing plant survival and production in many and very diverse plants of high economic interest in scientific trials and at pilot scale (Gianinazzi et al. 2002), However, the application and large-scale inoculation of HMA has always been paralyzed by i) the difficulty in obtaining a pure inoculum, light, free of contaminants and with high density of infective propagules; ii) the very variable results obtained with the mycorrhizal inoculants hitherto used, produced by a "conventional" system (see next paragraph), and whose application must be carried out in relatively mature and already pre-acclimatized plants. In fact, the absence in the market of aseptic mycorrhizal inoculants makes it impossible for the plant to leave mycorrhized immediately after in vitro rooting and prior to acclimatization, in the case of micropropagated plants.

The few HMA-based inoculants that exist today in the market come, in the vast majority, from fungus-plant crops produced in a greenhouse or culture chamber, and use various types of "dirty" substrates such as soil, sand, attapulgite as support. , sepiolite, vermiculite, etc. The production of HMA inoculants through these methods encounters three fundamental problems: i) under these conditions (open cultures) it is practically impossible to control the presence in the substrate of Accompanying microorganisms (generally unwanted) in addition to AMF, which makes validation and quality controls of these products difficult; ii) the manipulation of substrates such as those mentioned above is cumbersome and laborious, requiring large areas for their preparation, high cost of transport in their commercialization and making it difficult to incorporate them massively in large-scale farms; and iii) by retaining part of the substrate in which they were produced (these are "raw" inoculums), the above-mentioned inoculants retain a large amount of the aforementioned accompanying microorganisms, so that their use for sterile mycorrhization is excluded , as it might be desirable in the case of the preparation of seedlings, and of plants obtained by massive production techniques (ie micropropagation of plants by in vitro culture). Precisely, the in vitro plant micropropagation technique currently represents the best alternative in the market for rapid cloning and mass and certified production of plants of fruit and vegetable interest. Based on the large-scale multiplication of plants within glass vessels containing sterile culture medium, the technique consists of five phases: multiplication, elongation, rooting, acclimatization and hardening, the first three phases in vitro and the next two phases being performed. ex vitro. As can be deduced from the above, at present the introduction of mycorrhizal inoculants in micropropagated plants can only be carried out in the acclimatization or hardening phase (that is, ex vitro; eg Estrada-Luna et al. 2000; Marín et al. 2003 ), since these inoculants come from non-sterile conditions that prevent their introduction into the previous in vitro phases. This supposes a great delay in the introduction of the mycorrhizae, and an added stress to the micropropagated plant, which in addition to having to adapt to the new and harsh external conditions, must support the carbon drainage that the HMA demands, initially important until that the symbiotic balance is established (Bago et al. 2000). It is necessary, therefore, to find technical solutions that allow mycorrhizing plants both at the beginning of the seedbed (production of ex vitro plants), or before the acclimatization phase (micropropagated plants, in vitro production), that is, to be able to design a sterile HMA inoculum that makes compatible mycorrhization and rooting. This would reduce stress and the consequent losses that occur during the acclimatization phase of the plants (they have been set at up to 60% in the case of micropropagates) to the natural environment. In addition, the formation of mycorrhizae at such an early age would involve the production of prepared ("vaccinated") plants to resist other types of biotic and abiotic stresses, with the consequent reduction in the use of phytosanitary products; and would minimize delays in the establishment of mycorrhiza, the main determinant of the high variability obtained in the field results, which depend largely on the percentage of success in the establishment of the symbiosis. Finally, the transplantation of pre-mycorrhized plants to the natural environment would make them more competitive when it comes to acquiring nutrients from the natural environment, with the consequent benefits in terms of reducing chemical fertilizer application and / or adaptation to poor or degraded soils. In 1975 a system of in vitro co-cultivation of plant roots and HMA was devised (Mosse and Hepper, 1975), later developed and patented (Mugnier and Mosse, 1987; FR-2528865). This system, currently known as "monoxenic culture of arbuscular mycorrhizae" (CMMA) (Williams, 1992; Bago, 1998), consists of co-cultivating in vitro sterile explant root conditions ("root organ cultures", ROC) , together with HMA propagules isolated from the ground and sterilized on the surface. Under these controlled conditions, HMA and ROC establish mycorrhizal symbiosis, resulting in a large number of fungal propagules (resistance spores, mycorrhized root pieces, extraradical mycelium) in vitro (Bago and Cano, 2005). This technique has been modified on several occasions (Declerck et al. 2005), giving rise to several more recent patents: obtaining HMA propagules in physical absence of the root using medium compartments (St-Arnaud et al, 1996; US- 5554530); Ia replication as a starting material of mycorrhized root pieces containing HMA vesicles (Strullu and Romand, 1987; FR-2568094); and the establishment of mycorrhizae in vitro using undifferentiated plant cells (plant calluses, WO2005000008). However, none of the aforementioned patents or products have been marketed or marketed herein under STERILE CONDITIONS, the production of HMA being in In vitro only a large-scale production system of HMA propagules that, in pre-sale processing, is diluted with non-sterile substrates. With this one of the fundamental advantages of these products is lost, which still cannot be applied under aseptic conditions in the nursery chain of plant production. The only attempt to mycorrhize micropropagated plants under in vitro conditions (Elmeskaoui et al. 1995) was carried out using a solid culture medium in which plants whose roots were wrapped in cellulose cylinders were superimposed, which hindered their growth and reduced importantly the contact of the root with the inoculant.

The present invention exploits the potential of the monoxenic culture by improving it to design an aseptic inoculant suitable for mycorrhising plants in the rooting phase, both in in vitro conditions (nursery production) and ex vitro (from planters or seedlings to farms and revegetation of degraded soils) . To do this, it uses a medium-sized, semi-solid, low-viscosity, medium-sized culture medium that facilitates the penetration of the root into the inoculant, in addition to enhancing the infectivity of AMF. The inoculum object of the present invention also has numerous advantages over commercial inocula already existing in the market, since its application is easier, faster and cleaner, in addition to allowing aseptic certification until the moment of application .

References:

Bago B. (1998) AM monoxenic cultures using tomato non-transformed roots.

Development and Function of the Mycelium of Arbuscular Mycorrhizal Fungí.

Method Manual Kling, M. (ed.). Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden, pp. 41-44.

Bago B., Cano C. (2005) Breaking myths on arbuscular mycorrhizas In vitro biology. In: In vitro biology of mycorrhizal symbiosis. Declerck S., Strullu

D. G., Fortín A. (eds). SoN Biology Series, Springer-Verlag VoI. 4. pp. 111-138

Bago B, Shachar-Hill Y, Pfeffer PE (2000). Coal metabolism and transport in arbuscular mycorrhizas. Plant Physiol 124: 949-957

Declerck S., Strullu D. G., Fortín A. (eds). (2005) Breaking myths on arbuscular mycorrhizas in vitro biology. In: In vitro biology of mycorrhizal symbiosis. Soil Biology Series, Springer-Verlag VoI. Four.

Elmeskaoui A, Damont JP, Poulin MJ, Piché Y, Desjardins Y (1995) A tripartite culture system for endomycorrhizal inoculation of micropropagated strawberry plantlets in vitro. Mycorrhiza 5: 313-319 Estrada-Luna AA, Davies FT, Egilla JN (2000) Mycorrhizal fungi enhancement of growth and gas Exchange of micropropagated guava (Psidium gujava L.) plantlets during ex vitro acclimatization and plant establishment. Mycorrhiza 10: 1-8

Gianinazzi S, Schüepp H (eds.) (1994) Impact of Arbuscular Mycorrhizas on

Sustainable Agriculture and Natural Ecosystems. Birkhauser-Verlag, Basel.

226 pp

Gianinazzi S, Schüepp H, Barea JM, Haselwandter K (2002) Mycorrhiza Technology: From Genes to Bioproducts -Achievements and Hurdles in

Arbuscular Mycorrhizal Research. 296 pp. Giovannetti M, Mosse B (1980) An evaluation of techniques for measuring vesicular-arbuscular mycorrhizal infection in roots. New Phytol 84: 489-500 Harwood, RR (1991) A history of sustainable agriculture. In: LaI, R and Pierce FJ (eds.) Soil Management for Sustainability. Soil and Water Conservation

Society of America, Ankeny, pp. 3-19 Jeffries P, Barea JM (2001) Arbuscular mycorrhiza -a key component of sustainable plant-soil ecosystems. In: The Mycota IX. Fungal Associations Hock B (ed.). Springer Verlag, Berlin, pp. 95-113

López-Gorgé, J (1991) Fixation and biological mobilization of nutrients: photosynthesis, nitrogen fixation, mycorrhizae. In: Fixation and Mobilization

Biological Nutrient (VoI. I). López-Gorge, J (ed.). CSIC-New Trends, Madrid, pp. XI-XII

Marin M, Mari A, lbarra M, García-Ferriz L (2003) Arbuscular mycorrhizal inoculation of micropropagated persimmon plantlets. J. Hort. Sci. Biotechnol. 78: 734-738.

Mosse B (1986) Mycorrhiza in a sustainable agriculture. Biol Agrie Hortic 3: 191 -

209 Mosse B, Hepper C (1975) Vesicular-arbuscular infections in root organ cultures.

Physiol Plant Pathol 5: 215-223 Mugnier J, Mosse B (1987) Vesicular-arbuscular infections in transformed root-inducing T-DNA roots grown axenically. Phytopathol 77: 1045-1050 Sieverding E, Barea JM (1991) Perspectives of the inoculation of plant production systems with mycorrhizal fungi VA. In: Fixation and Biological Mobilization of Nutrients (VoI. II). Olivares J, Barea JM (eds.). CSIC-New Trends, Madrid, pp. 221-24

St-Amaud M, Hamel C, Vimard B, Carón M, Fortín JA (1996) Enhanced hyphal growth and spore production of the arbuscular mycorrhizal fungus Glomus intraradices in an in vitro system in the absence of host roots. Mycol Res 100:

328-332 Smith SE, Read DJ (1997) Mycorrhizal Symbiosis. Academic Press, London, 605

PP

Strullu DG, Romand C (1987) Axénique de vesicules culture solemn from the random and in-house re-association of tomato rabbits. CR Acad Sci Paris 305: 15-19 Williams PG (1992) Axenic culture of arbuscular mycorrhizal fungi. Methods Mic EXPLANATION OF THE INVENTION

As mentioned above, it would be convenient to have a sterile HMA inoculum to effectively mycorrhize plant, which remains aseptic for at least the time of its application. The present invention describes an inoculant of mycorrhization produced in vitro of semi-solid consistency, with a high content of HMA propagules, with high infectivity and colonization capacity of the roots, to mycorrhize plant both in vitro and ex vitro, which guarantees its aseptic conditions until the moment of its application.

Therefore, an object of the present invention is an inoculant of mycorrhization produced in vitro of semi-solid consistency and low viscosity (less than 2700 centipoise / minute). Said inoculant can originate from monoxenic or polygenic cultures of mycorrhizae that is maintained in aseptic conditions at least until the same moment of its application. The inoculant comprises at least one arbuscular endomicorrhizal fungus (AMF), belonging to different families of fungi (Glomeraceae, Gigasporaceae, Acaulosporaceae, Archaeosporaceae, Paraglomaceae, etc.), and may also comprise a combination of two or more endomicorrhizal fungi and other beneficial microorganisms of the ground. Said mycorrhizal fungi may come from areas with ecophysiological characteristics similar to that of the final application, or may have been specifically isolated from the same area in which they will be reintroduced.

The average content of AMF propagules (spores, mycorrhized roots and extraradical mycelium) is very high, at least 500 propagules / ml_, preferably between 2000 and 5000 propagules / ml_, with high infectivity (up to 20% of MA colonization (mycorrhizae arbuscular) in the roots inoculated two weeks after application).

The inoculant object of the invention can be presented in the form of dehydrated powder and mixed with inert substrates usually used in nursery cultivation of plants. The inoculant can also be a means aseptic mycorrhization that has one, two or more phases of different characteristics.

Another aspect of the present invention is the use of the inoculant described above to mycorrhize plants in vitro (micropropagated plants during or immediately after their rooting phase, and before the acclimatization phase), and ex vitro (micropropagated plants during the phase of acclimatization, and seedlings, alveoli, seedlings, nursery plants or any other plant obtainable from conventional nursery techniques).

Another aspect of the invention includes the application of the inoculum together with the irrigation water, at the same time as the sowing of seeds, and / or after the sowing of seeds by drip irrigation, irrigation trucks or sprinklers. Likewise, the application of the inoculum can be carried out both in standard or hydroponic seedbeds, as in agricultural farms.

The plants susceptible to being mycorrhized with the present inoculum are, among others, woody, shrubby plants, of fruit and vegetable interest, ornamental, flowers, vines, aromatic plants, medicinal plants, legumes, etc.

Another aspect of the present invention is the use of the inoculant for recovery and restoration by means of vegetative cover of degraded soils, and for the revegetation of altered areas subjected to biotic and abiotic stresses, highlighting as soils and degraded areas, among others, road slopes, mining areas, burned areas, desertified areas, contaminated soils, infested soils, soils degraded by excess salts, calcareous soils, extreme pH soils, etc.

BRIEF DESCRIPTION OF THE CONTENT OF THE FIGURES Fig. 1. Arbuscular mycorrhization in olive roots (left) and peach tree (right) micropropagated after two and a half months of inoculation with the object of the invention. Fig. 2. a. Comparison between micropropagated vines after six months of cultivation in a limestone-sandy agricultural soil in the absence (control) and presence (+ inoculant) of the inoculant object of the invention, b. Arbuscular mycorrhization in micropropagated vine roots after a year and a half of their inoculation with the object of the invention. Left, general view of the HMA colonization. Right, detail of symbiotic fungal structures.

Fig. 3. Comparison between lettuce plants obtained from seed and cultivated for three months in an agricultural soil in the absence (control) and presence (+ inoculant) of the inoculant object of the invention. Fig. 4. Comparison between leek plants obtained from seed and cultivated for three months in an agricultural soil in absence (control) and presence (+ inoculant) of the inoculant object of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The object of the present invention is an inoculant of mycorrhization produced in vito, of semi-solid consistency, with a viscosity of less than 2700 centipoise / minute, preferably between 1800 and 100 centipoise / minute, and more preferably between 800 and 200 centipoise / minute. . Said reduced viscosity is one of the most interesting novelties of the invention, since it increases the viability and infectivity of the inoculant, facilitating its final application in its various forms (gel, irrigation water, aseptic mycorrhization medium for micropropagated plants or diluted dry powder with various inert substrates). The semi-solid consistency and the viscosity of the inoculant are fundamental improvements with respect to the solid media (viscosity greater than 2700 c / minute) originally described for mycorrhizal monoxenic cultures, since this modification facilitates the penetration of the roots in the inoculant , leading to greater and more immediate contact of these with the HMA. Also, the high water content of the gel-type inoculant enhances the germination of seeds included therein, accelerating the HMA-root contact, and therefore also accelerating the establishment of mycorrhiza.

The inoculant object of the invention can originate from a monoxenic or polygenic mycorrhizal culture, and is maintained in aseptic conditions at least until the same moment of its application, which is another of the fundamental novelties of this invention. The maintenance of said aseptic conditions throughout the production and even the application by the user makes this inoculant superior to all currently existing at the level of purity, certification, infectivity and validation.

The mycorrhizal inoculant object of the invention comprises at least one strain of endomicorrhizal fungus (preferably an arbuscular endomicorrhizal fungus, HMA) that may belong to the Phylum Glomeromycota, preferably to the Glomaceae Families, Acaulosporaceae, Archaeosporaceae, Paraglomaceae or Gigasporaceae, and in particular to Genus Glomus, Entrophospora, Acaulospora, Archaeospora, Paraglomus, Gigaspora, Pacispora or Scutellospora. In a particular embodiment of the invention the endomicorrhizal fungus belongs to the genus Glomus, and in another particular embodiment of the invention the endomicorrhizal fungus belongs to the genus Gigaspora. In another particular embodiment of the invention, the following strains, in isolation or in variable combinations, may be part of the inoculant: MUCL 44632, MUCL 44633, CIMA10, CIMA12, CIMA13, CIMA07.

Likewise, the mycorrhization inoculant object of the invention can comprise a combination of two or more AMF, preferably within the families, genera and strains mentioned above, as well as in a combination of one or more endomicorrhizal fungi and other beneficial soil microorganisms.

It is important to note that the fungus or combination of mycorrhizal fungi present in said inoculant come from areas of ecophysiological characteristics similar to that of the final application, or have been specifically isolated from the same area in which they are to be reintroduced, which is another of the novelties and strengths of the invention object of this patent. Indeed, numerous scientific studies have shown that the efficacy of microbial strains in general, and of AMF in particular

The average content of AMF propagules (spores, mycorrhized roots and extraradial mycelium) of the inoculant object of the invention is at least 500 propagules / ml_, and preferably between 2000 and 5000 propagules / ml_, which represents a substantial increase with respect to other existing mycorrhizal inoculants. Another of the novel aspects of the present invention consists in the high infectivity of the inoculant object of this patent, which is always greater than that of dry mycorrhizal inoculants in any of its formulations (eg vermiculite, sepiolite, terragreen, perlite, sand, peat , clay minerals, etc.), presenting the roots entry points and up to 30% of colonization MA (arbuscular mycorrhizae) after two weeks of application of the inoculant. Obtaining said percentage of mycorrhization in such short times implies a substantial improvement granted by the inoculant object of the invention with respect to other HMA inoculants, and ensures the functional establishment of the mycorrhizae in the plant, and therefore obtaining the benefits that is derived from it, when said plant is transplanted to its final substrate.

The aseptic mycorrhization inoculant object of the invention can also be presented in the form of dehydrated powder, coming from the mixture of the inoculant-gel described above, with any inert substrate usually used in plant nursery cultivation. In a particular embodiment of the invention, the inoculant was mixed with vermiculite in an inoculant ratio: inert substrate of 1: 2 Likewise objects of the present invention are any mycorrhization composition and aseptic mycorrhization medium comprising an inoculant with the characteristics described above.

Said aseptic mycorrhization medium may have more than one phase, of which at least one is the inoculant object of the invention. In the case of consisting of two or more phases, the phase that contains the inoculant is under another, consisting either of a gelsed medium, or an inert substrate of the vermiculite, sepiolite, terragreen, perlite, sand, peat, soil type , minerals of Ia clay, etc. The reason for superimposing a phase on the inoculant object of the invention is based on several reasons, of which three are fundamental: i) the saving of inoculant that this implies; ii) since the AMF present in the inoculant phase is alive, during the mycorrhization process its external mycelium will grow and colonize the upper phase, with hyphae in full growth and eager to find a new root to mycorrhize, thus increasing even more the potential infective of the inoculant; and iii) the upper phase would act as a kind of "anti-stress buffer" allowing the roots to acclimatize to their new culture medium before coming into contact with the AMF in the inoculating phase. Another use of the present invention is the use of the inoculant, the mycorrhization composition and the aseptic mycorrhization medium to mycorrhize plants, whether micropropagated or not, in various ways:

• In vitro, during or immediately after the rooting phase, and before the acclimatization phase of the plants.

• Ex vitro, during the acclimatization phase of micropropagated plants, seedlings, nursery plants or any other plant obtainable from conventional nursery techniques.

• As an aseptic mycorrhization medium, in which case it is presented in sterile bottles of those commonly used in the micropropagation of plants. The mycorrhization procedure by aseptic means is characterized in that the roots of the plants are embedded in the mycorrhization medium. The presentation of this aseptic means of mycorrhization in sterile bottles allows its use within the plant nursery production chain, with the consequent benefit, in terms of minimum cost increase, by the nurseryman. In addition, the high potential of the aseptic mycorrhization medium allows its reuse under sterile conditions for at least twice successive. The inoculant object of the invention can be used for inclusion in seedbeds, alveoli, pots, containers, etc. in which they are going to cultivate any type of plant, preferably in its more youthful stages. Its application can be carried out by means of the application of individual aliquots of inoculant, preferably less than 2 ml_, and more preferably less than 1 ml_, to the substrate where the seed or nursery seedling is placed, said substrate being any of those used usually in nurseries, for example peat, sand, soil, vermiculite, perlite, sepiolite, terragreen, clay minerals and their derivatives, and mixtures of said substrates in varying proportions. The inoculant object of the invention can also be applied by means of its distribution by irrigation water, or by the brief immersion of the roots of the plants in it before its transplantation to the seedbed.

It should be noted that as plants susceptible to being mycorrhized with the inoculant object of the invention are, among others: woody, shrubby plants, of fruit and vegetable interest, ornamentals, flowers, vines, aromatic plants, medicinal plants, legumes, etc. The use of the inoculant object of the invention in the aforementioned forms will promote the early establishment of the arbuscular mycorrhizal symbiosis, with the consequent benefit in terms of viability, survival, resistance to biotic and abiotic stresses, nutritional improvement, adaptability, productivity, etc. presented by mycorrhized plants with respect to those that are not.

The inoculant object of the invention can also be used to mycorrhize plants in agricultural holdings, in which case its application can be carried out: i) at the same time as the sowing of seeds and mixed with them in the application hopper; ii) after sowing seeds by drip irrigation, irrigation trucks or sprinklers; iii) by planting pre-mycorrhized seedlings from standard or hydroponic seedlings in which the inoculant object of the invention would have been previously included, as described; iv) by means of the brief immersion of the roots of the plants in the inoculant object of the invention before their transplantation to field. The application of the inoculant will preferably be carried out in a proportion not less than 50 propagules per seed or seedling, and more preferably in a proportion not less than 100 propagules per seed or seedling. The inoculant object of the invention can also be applied as an enhancer of the reestablishment (recovery and restoration) of the vegetation cover of degraded soils, and for the revegetation of altered areas subject to biotic and abiotic stresses. In this sense, the revegetar soils can be, among others, roadsides, mining areas, burned areas, desertified areas, contaminated soils, infested soils, soils degraded by excess salts, calcareous soils, extreme pH soils, etc. The inoculation of plants with HMA isolates originating from the soils to be recovered, and their reintroduction in those same soils, ensures the maintenance of their biodiversity, as well as their physico-chemical conditions, favoring the reinstatement of the own plant cover of the soil. area in question

Likewise, it is another object of the present invention the mycorrhization procedures, in vitro and ex vitro, that use at least one inoculant, mycorrhization composition and aseptic mycorrhization medium objects also objects of the present invention.

In order to verify the effects of the object of the invention described above, various tests were performed applying the inoculant on seeds and micropropagated plants under in vitro and ex vitro conditions. The results are shown in the following examples.

EXAMPLES OF EMBODIMENT OF THE INVENTION

EXAMPLE 1. Rapid mycorrhization of plants under sterile conditions {in vitro)

This test is intended to confirm the infectivity of the inoculant object of the invention under aseptic conditions. Under sterile conditions, twenty pregerminated seedlings (from sterilized surface seeds and germinated in vitro) of clover and tomato with incipient radicles were placed in in vitro culture bottles containing 100ml of the aseptic inoculant object of the invention ("mycorrhization medium""), produced according to the described technique, so that the radicles were fully embedded in it. The inoculant was previously analyzed by direct stereomicroscopic counting according to the "gridline intersect method" of Giovanetti and Mosse (1980, with modifications), resulting in its average content of HMA propagules per ml of product in 935 ¹ ZS spores, 637 ¹ Colonized root fragments, and> 1000 fragments of external mycelium, which together assumes> 2500 HMA propagules / mL. Clover seedlings were kept in the jars for two weeks, at 24 ° C and 12 h of sunlight, after which the type and percentage of colonization MA was analyzed. After two weeks of in vitro culture, an MA colonization greater than 30% was found, said colonization being predominantly young and arbuscular, with some vesicle in the more mature areas of the root. The tomato seedlings were left in the jars for three weeks, at 24 ° C and 12 h of sunlight, after which they were acclimatized ex vitro in a sterile mixture of agricultural soil: quartz sand (1: 9 v: v), staying like this for about a month. The survival at acclimatization was 100%, and the percentage of MA colonization greater than 50%. The percentages of mycorrhization obtained with the two species used ensure the effectiveness of the inoculant object of the invention for the rapid establishment of mycorrhizal symbiosis, with the consequent benefit to the plant.

EXAMPLE 2. Ex vitro mycorrhization of micropropagated plants In clean conditions, 20 commercial alveoli of about 5OmI capacity with low fertilization peat were prepared to which micropropagated olive and peach seedlings were transplanted, ready to begin their acclimatization phase. To the incipient radicles of each plant 1 ml of the aseptic inoculant object of the invention was added so that they were totally wetted by it. Said inoculum has the same characteristics as that used in Example 1. The seedlings were grown for a month in an acclimatization tunnel, and subsequently (a month and a half) in greenhouse conditions, with drip irrigation. At the end of the test, growth parameters (fresh weight, height) as well as the percentage and type of HMA mycorrhization were evaluated (Fig. 2), and compared with their corresponding control plants (not inoculated). Table 2 shows how mycorrhized plants showed greater growth than controls. Likewise, 80 pots of about 20 Ohm capacity were prepared under clean conditions with a sterile mixture of agricultural soil: quartz sand (1: 9 v: v) added with a slow-release granulated fertilizer (Osmocote, 1g / l) . To these pots micropropagated vineyard seedlings were transplanted, ready to begin their acclimatization phase. To the incipient radicles of each plant 1 ml of the aseptic inoculant object of the invention was added so that they were totally wetted by it. The seedlings were grown for six months in a plant culture chamber (24 ° C, 12 light hours), and subsequently transplanted into 2kg pots containing a sandy agricultural soil, with a COsCa content of not less than 20%. The plants were grown for six months in the open, with a water regime of three irrigations per week. One year after the start of this trial, which is still ongoing, the evaluation of non-destructive growth parameters (height) was performed, as well as a partial sampling of roots for the evaluation of the percentage and type of HMA mycorrhization, and its comparison with the control plants (without inoculation). The results are shown in Figure 3 and Table 1. It is important to note that the differences obtained in terms of physiological parameters for the vineyard are maintained after six months of cultivation in field conditions, which reinforces the consistency of the data obtained for olive trees and peach tree and indicates the prevalence of the beneficial effects of the inoculant object of the invention in real agronomic conditions.

Table 1. Average and maximum increase in the growth parameters of mycorrhized plants with respect to control plants and percentage of mycorrhizal colonization.

OLIVE:

PEACH TREE:

EXAMPLE 3. Ex vitro mycorrhization of seeds

Under clean conditions, 30 pots of 30OmI capacity were prepared with a sterile mixture of agricultural soil: quartz sand (1: 9 v: v) added with a slow-release granulated fertilizer (Osmocote, 1g / l). Approximately 6cm from the surface, leek and lettuce seeds were sown, to which 1 ml of the aseptic inoculant object of the invention was added per pot, so that the seeds were fully included in it. The inoculant used is the same as in examples 1 and 2. After germination, the number of seedlings was reduced to one per pot. The plants were grown for two and a half months in greenhouse conditions, with a water regime of three irrigations per week. At the end of the test, plant growth parameters (fresh weight, diameter, height) were evaluated, as well as the percentage and type of HMA mycorrhization, and they were compared with their corresponding control plants (not inoculated). The results appear in Figures 4 and 5 and in Table 2, where it can be seen that mycorrhized plants showed greater growth than controls. Table 2. Average and maximum increase in the growth parameters of mycorrhized plants with respect to control plants and percentage of mycorrhizal colonization.

LETTUCE:

Claims

1 - Inoculant of mycorrhization produced in vitro, characterized in that its consistency is semi-solid, with a viscosity of less than 2700 centipoise / minute, preferably between 1800 and 100 centipoise / minute, and more preferably between 800 and 200 centipoise / minute.

2- Mycorrhizal inoculant according to claim 1, characterized in that said inoculant originates from a mycorrhizal monoxenic culture.

3- Mycorrhizal inoculant according to claim 1 characterized in that said inoculant originates from a mycorrhizal polygenic culture.

4- Mycorrhization inoculant according to claims 1 to 3, characterized in that said inoculant is maintained in aseptic conditions at least until the same moment of its application.

5- Mycorrhizal inoculant according to claims 1 to 4, characterized in that said inoculant comprises at least one endomicorrhizal fungus, preferably an arbuscular endomicorrhizal fungus (AMF).

6- Mycorrhizal inoculant according to claim 5 characterized in that said endomicorrhizal fungus belongs to a single strain, preferably an arbuscular endomicorrhizal fungus strain. 7-mycorrhizal lnoculant according to any of the preceding claims characterized in that the endomicorrhizal fungus belongs to the Phylum Glomeromycota, preferably to the Glomeromycetes Class, and more preferably to the Glomeraceae Family.

8-mycorrhizal lnoculant according to claim 7 characterized in that the endomicorrhizal fungus belongs to the genus Glomus, preferably to the species G. intraradices

9-mycorrhizal lnoculant according to claim 8 characterized in that the endomicorrhizal fungus is selected from the following strains: MUCL 44633, CIMA10, CIMA12, CIMA13, CIMA07. 10-mycorrhizal lnoculant according to any one of claims 1 to 6, characterized in that the endomicorrhizal fungus belongs to the Gigasporaceae Family, preferably to the genus Gigaspora, and more preferably to the species G. margarita 11 -Micorrization inoculant according to claim 10 characterized in that the endomicorrhizal fungus of the G. margarita species belongs to the strain MUCL 44632.

12-mycorrhizal lnoculant according to any of claims 1 to 6, characterized in that the endomicorrhizal fungus belongs, among others, to one of the following Families: Glomaceae, Acaulosporaceae, Archaeosporaceae, Paraglomaceae or Gigasporaceae, and preferably to the Glomus genera, Entrophospora, Acaulospora, Archaeospora, Paraglomus, Gigaspora, Pacispora or Scutellospora.

13-mycorrhizal lnoculant according to claims 1 to 4, characterized in that said inoculant comprises a combination of two or more endomicorrhizal fungi, preferably arbuscular endomicorrhizal fungi of those cited in claims 1 to 12.

14-mycorrhizal lnoculant according to any of claims 1 to 13 characterized in that it comprises a combination of one or more endomicorrhizal fungi and other beneficial soil microorganisms.

15 - Mycorrhizal inoculant according to any of claims 1-14, characterized in that the fungus or combination of mycorrhizal fungi present in said inoculant comes from areas of ecophysiological characteristics similar to that of final application, or have been specifically isolated from the same area in Ia that will be reintroduced.

16 - Mycorrhizal inoculant according to any one of claims 1 to 15, characterized in that its average content of AMF propagules (spores, mycorrhized roots and extraradical mycelium) is at least 500 propagules / ml, preferably between 2000 and 5000 propagules. ml_. 17- Mycorrhizal inoculant according to any of claims 1 to 16, characterized in that its infectivity is always greater than that of a dry mycorrhizal inoculant in any of its formulations (eg vermiculite, sepiolite, terragreen, perlite, sand, peat, minerals of clay, etc.), presenting entry points and up to 30% of colonization MA (arbuscular mycorrhizae) in the inoculated roots two weeks after its application.

18- Aseptic mycorrhizal inoculant characterized in that said inoculant is presented in the form of dehydrated powder, from the mixture of the inoculant described in claims 1-17 and inert substrates commonly used in plant nursery culture.

19- Aseptic mycorrhization inoculant according to claim 18 characterized in that the inert substrate is vermiculite and the proportion of the mixture (inoculant: vermiculite) is at least (1: 2).

20 - Mycorrhization composition characterized in that it comprises at least one inoculant according to any one of claims 1 to 19.

21- Aseptic mycorrhization medium characterized in that it comprises at least one inoculant according to any one of claims 1 to 17.

22- Aseptic mycorrhization medium characterized in that it comprises two or more phases of which at least one of them is an inoculant according to any one of claims 1 to 17.

23- Aseptic mycorrhization medium according to claim 22, characterized in that the phase containing the inoculant is under another phase consisting of a gelsed medium or an inert substrate.

24- Aseptic mycorrhization medium according to claim 23, characterized in that the inert substrate is of the vermiculite, sepiolite, terragreen, perlite, sand, peat, soil, clay minerals, etc. type.

25- Use of an inoculant according to claims 1 to 20 to mycorrhize plants in vitro. 26- Use of an inoculant according to claim 25 for in vitro mycorrhization of micropropagated plants during or immediately after their rooting phase, and before the acclimatization phase.

27- Use of a mycorrhization medium according to claims 21 to 24 to micorrizar plants in vitro.

28- Use of a mycorrhization medium according to claim 27 to in vitro mycorrhize micropropagated plants during or immediately after their rooting phase, and before the acclimatization phase.

29- Use of an inoculant according to claims 1 to 20 to mycorrhize ex vitro plants.

30- Use of an inoculant according to claim 29 to mycorrhize micropropagated plants during the acclimatization phase.

31- Use of an inoculant according to claim 29 to mycorrhize seedlings, alveoli, seedlings, nursery plants or any other plant obtainable from conventional nursery techniques.

32-Use of an inoculant according to claims 1 to 20, characterized in that said inoculant is applied together with the irrigation water.

33-Use of an inoculant according to any of claims 1 to 20, characterized in that said inoculant is applied in agricultural holdings. 34-Use of an inoculant according to claim 33 characterized in that the application of said inoculant is carried out at the same time as the sowing of seeds and mixed with them in the application hopper.

35-Use of an inoculant according to claim 33 characterized in that the application of said inoculant is carried out after seed sowing by drip irrigation, irrigation trucks or sprinklers.

36-Use of an inoculant according to any one of claims 1 to 20, characterized in that the application of said inoculant is carried out in both standard and hydroponic seedbeds. 37. Use of an inoculant according to claims 34 to 36 characterized in that the inoculant is preferably applied in a proportion not less than 50 propagules per seed or seedling, and more preferably in a proportion not less than 100 propagules per seed or seedling. 38- Use of an inoculant according to claims 25 to 37, characterized in that the plants that are mycorrhized are, among others, woody, shrubby plants, of fruit and vegetable interest, ornamental, flowers, vines, aromatic plants, medicinal plants, legumes, etc.

39- Use of an inoculant according to claims from Ia 25 to Ia 38 for recovery and restoration by vegetation cover of degraded soils, and for the revegetation of altered areas subjected to biotic and abiotic stresses.

40- Use of an inoculant according to claim 39, characterized in that the soils and degraded areas that are treated are, among others, roadsides, mining areas, burned areas, desertified areas, contaminated soils, infested soils, degraded soils by excess salts. , calcareous soils, extreme pH soils, etc.

41- In vito mycorrhization procedure characterized in that at least one inoculant or aseptic mycorrhization medium according to claims 1 to 24 is used.

42- Ex vitro mycorrhization procedure characterized in that at least one inoculant according to claims 1 to 20 is applied.

43 - Mycorrhization method according to claim 42, characterized in that an aliquot of the inoculant, preferably less than 2 ml_, and more preferably less than 1 ml_, is added to the substrate where the seed or nursery plant is placed, said substrate being any substrate commonly used in nurseries, for example peat, sand, soil, vermiculite, perlite, sepiolite, terragreen, clay minerals and their derivatives, and mixtures of said substrates in varying proportions. 44- In vitro mycorrhization procedure characterized in that the roots of the plants are embedded in a mycorrhization medium according to claims from 21 to 24.

45- Ex vitro mycorrhization procedure characterized in that the roots of the plants are briefly immersed in a mycorrhization medium according to claims 1 to Ia 20, before being transplanted to a seedbed or to its definitive medium.

46- Ex vitro mycorrhization procedure characterized in that an inoculant according to claims 1 to Ia 20 is applied, directly to the soil of the agricultural holding according to claims from 32 to 37.